Steven F. Ziegler

: Regulatory T cells (Tregs) prevent autoimmunity through suppressive functions largely programmed by the transcription factor FOXP3. Healthy humans express approximately equivalent levels of two major alternatively spliced isoforms of FOXP3: a full-length version containing all coding exons (FOXP3-FL) and a version lacking exon 2 (FOXP3-ΔE2). However, sole FOXP3-ΔE2 expression causes lethal IPEX syndrome, and the FOXP3-ΔE2 isoform is elevated in several autoimmune diseases. These observations strongly suggest defects in suppression by FOXP3-ΔE2 Tregs which we investigated here using Foxp3-ΔE2 mice. In an influenza virus infection model, Foxp3-ΔE2 mice had an increased magnitude of the CD8 ⁺ T cell response during acute and memory formation phases of infection. Transcriptomic and chromatin accessibility analyses of homeostatic Foxp3-ΔE2 Tregs revealed impaired Treg programming, including reduced expression of inhibitory molecules such as Il2ra and chemokine receptors. Decreased cell surface CD25 expression on Foxp3-ΔE2 Tregs was associated with reduced IL-2 responsiveness in Foxp3-ΔE2 Tregs and, reciprocally, increased IL-2 responsiveness in CD8 ⁺ T cells from Foxp3-ΔE2 mice. Additionally, altered chemokine receptor expression resulted in diminished localization of Foxp3-ΔE2 Tregs to the T cell zone of the inflamed lymph node. Thus, Treg programming by the Foxp3-ΔE2 isoform impairs suppressive function, resulting in failure to restrain CD8 ⁺ T cells and aberrant immune responses. One Sentence Summary: Foxp3-ΔE2 expressing regulatory T cells have altered cellular programming which impairs their IL-2 sink function and co-localization with conventional T cells during priming, enhancing CD8 ⁺ T cell responses.

Host factors influencing susceptibility to rhinovirus-induced asthma exacerbations remain poorly characterized. Using organotypic bronchial epithelial cultures from well-characterized children with asthma and healthy children, this study investigated viral load kinetics and resultant host responses by bulk and single-cell transcriptomics and targeted protein analyses. Bronchial epithelium from exacerbation-prone children exhibited greater rhinovirus replication and a cascade of exaggerated downstream interferon (IFN), inflammatory, epithelial stress, and remodeling responses. These transcriptional patterns were confirmed and further refined using single-cell transcriptomics, revealing cell type–specific contributions — particularly from non-ciliated cell populations including secretory immune response, tuft, and basal cells. We observed that these post-infection differences were associated with lower pre-infection IFN-stimulated gene (ISG) expression and protein levels of the ISG CXCL10. Prophylactic IFN-β treatment reduced viral replication and normalized downstream responses, supporting low baseline (pre-infection) IFN tone as a modifiable causal determinant of host susceptibility to adverse rhinovirus-induced responses in exacerbation-prone children with asthma.

Thymic stromal lymphopoietin (TSLP) is an alarmin cytokine possessing a plethora of pleiotropic properties. Human and mouse TSLP exerts their activity via a heterodimeric complex composed of TSLP receptor (TSLPR) chain and IL-7Rα. TSLP is predominantly expressed by epithelial cells and keratinocytes but can also be produced by several immune cells and some cancers. TSLP activates a plethora of immune cells implicated in inflammation, angiogenesis and tumorigenesis. In addition to its role in barrier immunity, recent studies have a role for TSLP in cancer development. This includes both human hematologic cancers and several solid tumors (largely carcinomas). The role of TSLP in human and experimental cancers has been the focus of several studies, with somewhat contradictory findings. In this Review, we will highlight recent advances in TSLP immunobiology in the context of human and experimental cancers. We will also discuss recent findings demonstrating that an anti-TSLP monoclonal antibody (mAb) can exert a protective effect in a mouse model of colorectal cancer. The recent approval of an anti-TSLP mAb for asthma treatment also emphasizes the urgent need for additional research on the role of TSLP, a Janus cytokine, in tumorigenesis.

Cutaneous allergen sensitization (CAS) underlies atopic dermatitis (AD) and leads to various allergic symptoms, including food allergy and anaphylaxis. IL-13 expression by T follicular helper T (TFH) has been reported to be involved in generating high-affinity IgE antibodies and causing systemic anaphylaxis.1, 2 However, the mechanisms by which IL-13 triggers IgE-mediated allergic responses remain poorly defined. In the present study, we elucidate the role of IL-13 in the CAS-mediated mechanism by which high-affinity IgE antibodies are produced when the same allergen is introduced at a distal site in the secondary sensitization. The CAS model system using mice lacking the cell lineage-specific IL-13 receptor (IL-13R) demonstrated that dendritic cells (DCs), but not T or B cells, are critical in the high-affinity IgE-mediated anaphylactic response. The IL-13 signal in type 2 conventional DCs (cDC2s) enhanced the expression of MHC class II and CD301b, which was essential for the recall of type 2 responses, inducing the production of high-affinity IgE antibodies. Similar IL-13R-expressing DCs were identified in allergic rhinitis and food allergy patients with a history of AD. These findings strongly suggest the importance of DC-specific IL-13 signaling in CAS-induced allergic reactions associated with the atopic march, which is common in human AD patients.

Thymic stromal lymphopoietin is an established pleotropic alarmin cytokine that is generated at barrier tissues to induce type 2 immune responses, but its role in regulating the diversity of B cells is poorly understood. Here, we will highlight the key findings that underpin our limited understanding of the role thymic stromal lymphopoietin in modulating different stages of B cell development. We will also provide an overview of how thymic stromal lymphopoietin drives B cell-mediated immune disease and how novel thymic stromal lymphopoietin-blocking biologics could be used to modulate B cell responses. Thymic stromal lymphopoietin is critical for the regulation, diversity, and longevity of humoral immunity.

Regulatory T cells (Tregs) are a specialized subset of CD4⁺ T cells essential for the maintenance of immune homeostasis and prevention of autoimmunity. Treg lineage and functions are programmed by the X-chromosome encoded transcription factor Forkhead box P3 (FOXP3). In humans, multiple FOXP3 isoforms are generated through alternative splicing. A full-length isoform containing all coding exons (FOXP3-FL) and a version lacking the second exon (FOXP3-ΔE2) are the predominant FOXP3 isoforms. Additionally, there are two minor isoforms lacking either exon 7 (FOXP3-ΔE7) and both exons 2 and 7 (FOXP3-ΔE2ΔE7). Although healthy humans express approximately equal levels of the FOXP3-FL and FOXP3-ΔE2 isoforms, sole expression of FOXP3-ΔE2 results in development of a systemic autoimmune disease that resembles immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. These clinical observations strongly suggest functional defects in suppression by Tregs programmed by the FOXP3-ΔE2 isoform. Work from the past two decades has provided phenotypic and functional evidence of differences between Tregs programmed by the FOXP3-FL, FOXP3-ΔE2, and FOXP3-ΔE7 isoforms. In this review, we discuss the discovery of the FOXP3 isoforms, differences in the phenotype and function of Tregs programmed by different FOXP3 isoforms, and the role that these isoforms are known to play in autoimmunity.

Group 1 innate lymphoid cells (ILC1s) are cytotoxic and interferon gamma-producing lymphocytes lacking antigen-specific receptors, which include ILC1s and natural killer (NK) cells. In mice, ILC1s differ from NK cells, as they develop independently of the NK-specifying transcription factor EOMES, while requiring the repressor ZFP683 (ZNF683 in humans) for tissue residency. Here we identify highly variable ILC1 subtypes across tissues through investigation of human ILC1 diversity by single-cell RNA sequencing and flow cytometry. The intestinal epithelium contained abundant mature EOMES⁻ ILC1s expressing PRDM1 rather than ZNF683, alongside a few immature TCF7⁺PRDM1⁻ ILC1s. Other tissues harbored NK cells expressing ZNF683 and EOMES transcripts; however, EOMES protein content was variable. These ZNF683⁺ NK cells are tissue-imprinted NK cells phenotypically resembling ILC1s. The tissue ILC1-NK spectrum also encompassed conventional NK cells and NK cells distinguished by PTGDS expression. These findings establish a foundation for evaluating phenotypic and functional changes within the NK-ILC1 spectrum in diseases.

The transcriptional and phenotypic characteristics that define alveolar monocyte and macrophage subsets in acute hypoxemic respiratory failure (AHRF) are poorly understood. Here, we apply CITE-seq (single-cell RNA-sequencing and cell-surface protein quantification) to bronchoalveolar lavage and blood specimens longitudinally collected from participants with AHRF to identify alveolar myeloid subsets, and then validate their identity in an external cohort using flow cytometry. We identify alveolar myeloid subsets with transcriptional profiles that differ from other lung diseases as well as several subsets with similar transcriptional profiles as reported in healthy participants (Metallothionein) or patients with COVID-19 (CD163/LGMN). We use information from CITE-seq to determine cell-surface proteins that distinguish transcriptional subsets (CD14, CD163, CD123, CD71, CD48, CD86 and CD44). In the external cohort, we find a higher proportion of CD163/LGMN alveolar macrophages are associated with mortality in AHRF. We report a parsimonious set of cell-surface proteins that distinguish alveolar myeloid subsets using scalable approaches that can be applied to clinical cohorts.

Rationale: Indirect airway hyperresponsiveness (AHR) is a highly specific feature of asthma, but the underlying mechanisms responsible for driving indirect AHR remain incompletely understood. Objectives: To identify differences in gene expression in epithelial brushings obtained from individuals with asthma who were characterized for indirect AHR in the form of exercise-induced bronchoconstriction (EIB). Methods: RNA-sequencing analysis was performed on epithelial brushings obtained from individuals with asthma with EIB (n = 11) and without EIB (n = 9). Differentially expressed genes (DEGs) between the groups were correlated with measures of airway physiology, sputum inflammatory markers, and airway wall immunopathology. On the basis of these relationships, we examined the effects of primary airway epithelial cells (AECs) and specific epithelial cell–derived cytokines on both mast cells (MCs) and eosinophils (EOS). Measurements and Main Results: We identified 120 DEGs in individuals with and without EIB. Network analyses suggested critical roles for IL-33–, IL-18–, and IFN-γ–related signaling among these DEGs. IL1RL1 expression was positively correlated with the density of MCs in the epithelial compartment, and IL1RL1, IL18R1, and IFNG were positively correlated with the density of intraepithelial EOS. Subsequent ex vivo modeling demonstrated that AECs promote sustained type 2 (T2) inflammation in MCs and enhance IL-33–induced T2 gene expression. Furthermore, EOS increase the expression of IFNG and IL13 in response to both IL-18 and IL-33 as well as exposure to AECs. Conclusions: Circuits involving epithelial interactions with MCs and EOS are closely associated with indirect AHR. Ex vivo modeling indicates that epithelial-dependent regulation of these innate cells may be critical in indirect AHR and modulating T2 and non-T2 inflammation in asthma.

The thymus is an intricate organ consisting of a diverse population of thymic epithelial cells (TECs). Cortical and medullary TECs and their subpopulations have distinct roles in coordinating the development and selection of functionally competent and self-tolerant T cells. Recent advances made in technologies such as single-cell RNA sequencing have made it possible to investigate and resolve the heterogeneity in TECs. These findings have provided further understanding of the molecular mechanisms regulating TEC function and expression of tissue-restricted Ags. In this brief review, we focus on the newly characterized subsets of TECs and their diversity in relation to their functions in supporting T cell development. We also discuss recent discoveries in expression of self-antigens in the context of TEC development as well as the cellular and molecular changes occurring during embryonic development to thymic involution.